Benz-indole and benzo-quinoline derivatives as prodrugs for tumor treatment

ABSTRACT

Compounds of the general formula (I) or (IA) in which X is H, Y is a leaving group, R 1  preferably being an aromatic DNA binding subunit are prodrug analogues of duocarmycin. The compounds are expected to be hydroxylated at the carbon atom to which C is joined, by cytochrome P450, in particular by CYP1B1, expressed at high levels in tumors. The prodrug is expected to be activated preferentially in tumor cells, which it will act as a DNA alkylating agent preventing cell division.

The present invention concerns aromatic oxidation/hydroxylation activated prodrugs, particularly anti-tumor prodrugs and those which are specifically activated by the oxidation/hydroxylation activities of the cytochrome P450 family of enzymes.

Many conventional cytotoxic drugs are known that can be used for therapeutic purposes. However, they typically suffer from the problem that they are generally cytotoxic and therefore may affect cells other than those that are required to be destroyed. This can be alleviated to some extent by the use of targeted drug delivery systems, for example direct injection to a site of tumorous tissue or, e.g. binding the cytotoxic agent to an antibody that specifically recognises an antigen displayed only on the cancer cell surface. Alternatively, electromagnetic radiation may be used to cause chemical alteration in an agent at a desired site such that it becomes cytotoxic. However, all of these techniques have, to a greater or lesser extent, certain limitations and disadvantages.

The compound (+)-CC-1065 and the duocarmycins are naturally occurring representatives of a class of DNA alkylating agents. The naturally occurring compounds consist of a DNA alkylating unit based upon a pyrrolo[3,2-e]indole core, with one or two sub units, conferring DNA binding capabilities. CC-1065 and duocarmycin A comprise a spirocyclic cyclopropane group responsible for the DNA alkylation properties. Duocarmycin B₂, C₂ and D₂ are believed to be precursors for cyclopropane actives, and comprise a substituted (by a leaving group) methyl group at the eight position on the dihydro pyrrole ring. CC-1065 has been synthesised by various routes, summarised by Boger et al in Chem. Rev. 1997, 97, 787-828.

In U.S. Pat. No. 4,413,132 the first synthesis of the left hand sub-unit of CC-1065 was described. The synthesis is based on a Winstein Ar-3′ alkylation in which the cyclopropane ring is introduced. In a previous step, the A ring (of the indole core) is introduced by reaction of an aniline with an α-thiomethylester using chemistry based on Gassman's Oxindole Synthesis. The aniline has a protected phenolic hydroxyl group ortho to the NH₂ group, which, in the final product, is believed to be crucial for DNA alkylation. CC-1065 has broad antitumor activity but is too toxic against normal cells to be clinically useful.

Boger et al (1997) op.cit. also describe various deep-seated structural modifications of the DNA-alkylation subunit, in which the pyrrolo ‘A’ ring is replaced by other aromatic ring structures. In one class of analogues the replacement ring is a benzene ring.

Attempts have been made to target the delivery of CC-1065 and analogues by conjugating the drug via the DNA binding subunit to polymers, or specific binding agents such as antibodies or biotin described in U.S. Pat. No. 5,843,937. Boger et al in Synthesis 1999 SI, 1505-1509 described prodrugs of 1,2,9,9a-tetrahydrocyclopropa(c)benz[e]indol-4-one, in which the cyclopropane ring-opened version of the compounds were derivatised by reaction of the phenolic group to form esters and carbamates.

In Tet. Letts. (1998) 39, 2227-2230 Boger et al., describe (the synthesis of a range of precursors for the alkylation subunits of duocarmycin and CC-1065 analogues having a deep-seated structural modifications of the alkylation subunit. One of the compounds synthesised is a benzodihydroindole derivative:

In J. Org. Chem. (2000), 65/13, 4101-4111 the corresponding ring closed indoline compound (CBI derivative) derived from that benzodihydroindole derivative was coupled to the DNA-binding subunit of CC-1065 and shown to have DNA alkylation activity. Analogues of the alkylation subunit precursor in which the benz moiety is substituted with methoxy or cyano were also synthesised. Similar compounds are described in WO-A-9745411 and WO-A-9732850. In '411 the benz moiety is substituted by cyano at the 7-position or the cyclopropane group may be difluoro substituted. In '850 the 7-methoxy CBI compounds are described.

In WO-A-9811101 the phenolic hydroxyl group in the B ring of CBI-type compounds is replaced by amino, nitro or thiol-based groups.

In J. Am. Chem. Soc. (1991), 113, 3980-3983 Boger et al describe a study to identify features of CC-1065 analogues contributing to the selectivity of the DNA-alkylation. The compounds tested in vitro had alkylating subunits based on 2,3-dihydroindole and included the 6-deshydroxy analogues. These were shown to have some DNA alkylating properties though at concentrations 10⁴ times higher than that of the 6-hydroxy compounds.

The present invention relates to precursors of analogues CC-1065, which do not have the hydroxyl group in the B ring of the alkylating sub unit, and which are hence substantially inactive as DNA alkylating agents themselves.

It has been reported (Murray, G. I. et al., 15 Jul. 1997, Cancer Research, 57 m 3026-3031 and WO-A-9712246) that the enzyme CYP1B1, a member of the cytochrome P450 (CYP) family of xenobiotic metabolising enzymes, is expressed at a high frequency in a range of human cancers, including cancers of the breast, colon, lung, oesophagus, skin, lymph node, brain and testes, and that it is not detectable in normal tissues. This led to the conclusion that the expression of cytochrome P450 isoforms in tumor cells provides a molecular target for the development of new antitumor drugs that could be selectively activated by the CYP enzymes in tumor cells, although no drug examples were given. A number of other CYP isoforms have been shown to be expressed in various tumors. Many of the CYP's expressed in tumors are mentioned in Patterson, L H et al, (1999) Anticancer Drug Des. 14(6), 473-486.

In WO-A-99/40056 prodrugs of styrene- and chalcone-derivatives are described. The respective hydroxylated forms of the prodrugs, formed in situ, are potent tyrosine kinase (TK) inhibitors. Inhibition of TK activity contributes to tumor inhibition and cell destruction. The prodrugs were shown to be activated by microsomal preparations expressing CYP1B1 enzyme, and to have cytotoxic activity against cell lines expressing the same enzyme, whilst having much lower cytotoxic activity against cell lines not expressing the enzyme.

The present invention is directed to a new class of prodrugs which are expected to be hydroxylated in situ by CYP enzymes, in particular enzymes expressed at high levels in tumors. In particular the prodrugs are believed to be metabolisable by CYP1B1 enzyme. Some of the compounds are new. The present invention relates to the first therapeutic use of a broad range of compounds, and their synthesis as well as intermediates used therein.

There is provided according to the first aspect of the invention the new use of a compound of the general formula I or IA or a salt thereof in the manufacture of a composition for use in a method of treatment by therapy of an animal:

in which,

X is H;

Y is a leaving group

R¹ is —Ar, NH₂, OR⁷ or R⁷;

R², R³, R⁴R⁵ and R⁶ are each independently selected from H, C₁₋₄ alkyl, —OH, C₁₋₄alkoxy, —CN, Cl, Br, I, —NO₂, —NH₂, —NHR¹⁶, —NR¹⁶ ₂, —N⁺R¹⁶ ₃, —NHCOR⁸, —COOH, CONHR⁹, —NHCOOR⁹ and —COOR⁹;

R⁷, R⁸ and R⁹ are independently selected from C₁₋₄alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl and a ligand;

Ar is selected from

in which

B is N or CR¹⁰;

R¹⁰ is selected from OH, C₁₋₄alkoxy, C₁₋₄ alkyl, —NO₂, —NH₂, —CN, Cl, Br, I, —NHCOR¹⁴, —COOH, —CONHR⁵, —NHCOOR¹⁵ and —COOR¹⁵ and H;

Z is O, S, —CH═CH— or NH;

the or each R¹¹ is selected from OH, C₁₋₄ alkoxy, C₁₋₄ alkyl, —NO₂, —NH₂, —NHR¹⁶, —NR¹⁶ ₂, —N⁺R¹⁶ ₃, —CN, Cl, Br, I, —NHCOR¹⁴, —COOH, —CONHR¹⁵, —NHCOOR¹⁵ and —COOR¹⁵;

n is an integer in the range 0 to 4;

R¹² is H, —COAr¹, —CONH₂, —COOH, —COOR¹⁵ or —COR¹⁵;

the or each R¹³ is selected from OH, C₁₋₄ , alkoxy, C₁₋₄ alkyl, —NO₂, —NH₂, —NHR⁶, —NR¹⁶ ₂, —N⁺R¹⁶ ₃, —CN, Cl, Br, I, —NHCOR¹⁴, —COOH, —CONHR¹⁵, —NHCOOR¹⁵ and —COOR¹⁵;

m is 0, 1 or 2;

R¹⁴ is selected from C₁₋₄ alkyl, optionally substituted phenyl, optionally substituted heteroaryl, C₇₋₁₂ aralkyl, Ar¹ and ligands;

R¹⁵ is selected from C₁₋₄ alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl optionally substituted heteroaryl and ligands;

the or each R¹⁶ is independently selected from C₁₋₄ alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl and optionally substituted heteroaryl; and

Ar¹ is selected from the same groups as Ar;

provided that no more than one group R¹¹ or R¹³ in any one ring includes a group Ar¹.

Ar¹ is preferably

The animal which is treated is generally a human, although the compounds may also have veternary use. The indication treated is generally cancer including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.

The tumor may, for instance, be defined as a tumor expressing high levels of CYP1B1.

In the invention, the leaving group Y is, for instance, in a group which has utility in nucleophilic substitution reactions. Suitable examples of leaving groups are —OCOOR¹⁷, —OCONHR¹⁸, Cl, Br, I, or —OSOOR¹⁹, in which R¹⁷, R¹⁸ and R¹⁹ are selected from C₁₋₄ alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl and optionally substituted heteroaryl. Most preferably the leaving group is a halogen atom, preferably chlorine.

Optional substituents in phenyl, aralkyl and heteroaryl groups are, for instance, C₁₋₄-alkyl, halogen, hydroxyl, C₁₋₄-alkoxy, —NH₂, —NHR¹⁶, —NR¹⁶ ₂, —N⁺R¹⁶ ₃, —NO₂, —CN, —COOH, —NHCOR⁴, —CONHR¹⁵, —NHCOOR⁵, —COOR¹⁵ etc.

In the present invention the term ligand includes a group having specific targeting characteristics, useful for instance in antibody or gene-directed enzyme prodrug-type environments. A ligand may be an oligopeptide, biotin, avidin or streptavidin, a polymeric group, an oligonucleotide or a protein. Preferably it has specific binding characteristics such as an antibody or fragment, an antigen, a sense or anti-sense oligo-nucleotide, or one of avidin, streptavidin and biotin, that is it is one component of a specific binding pair. Alternatively it may be a group designed for passive targeting, such as a polymeric group, or a group designed to prolong the stability or reduce immunogenicity such as a hydrophilic group. U.S. Pat. No. 5,843,937 discloses suitable ligands for conjugating to these types of actives and methods for carrying out the conjugation.

In a pharmaceutically active compound R¹ is other than OR⁷.

In general, for optimised DNA binding ability, the group R¹ in a compound of the general formula I is a group Ar. Often the compound may include two aromatic groups joined to one another. In such compounds, one of the groups R¹¹ of the Ar group, or the group R¹², as the case may be, is a group Ar¹. Whilst for some compounds it may be desirable for three or more such aromatic groups to be linked, it is preferred that there is one group Ar and one group Ar¹. Thus in a group Ar¹ which is a pyrrolo-dihydroindole type of group, the group R¹² should be other than a group —COAr¹. In a group Ar¹ which is one of the other types of group there should preferably either be no substituents R¹¹ or R¹³, as the case may be, or, if there are any substituents, no such substituents should include a group Ar¹.

According to one embodiment of the invention, the substituent Ar is a group

In such groups Ar¹, B is preferably CR¹⁰. R¹⁰ is preferably H. The definition of Z is preferably NH, although furan (Z═O) and thiophene (Z═S) analogues had been generated for conjugation to DNA alkylating units and may have useful DNA binding characteristics. Similarly, in a group Ar¹, the groups B and Z are selected amongst the same preferable groups. Preferably n is at least 1 and one of the groups R¹¹ is —NHCOAr¹. In this embodiment Ar¹ is preferably a group

in which B and Z are the same as in Ar.

In another embodiment the substituent Ar is a group

Preferably R¹² in Ar is other than —COOR¹⁵, more preferably it is a group —COAr¹ in which Ar¹ preferably is the same type of group.

In both groups Ar and Ar¹, m in the indole type group is preferably zero.

In Ar and Ar¹, there may be several substituents R¹¹. Most preferably such substituents are selected amongst C₁₋₄-alkoxy groups.

In compounds of the formula I, the core indole ring of the DNA alkylating sub-unit is preferably unsubstituted in the benzene ring (R² is hydrogen), whilst the benz ring may be unsubstituted (R³, R⁴, R⁵ and R⁶ are all hydrogen), or one or more of R³ to R⁶ represents a cyano group, an alkoxy group, a group —COOR¹⁰, or a C₁₋₄-alkyl group (preferably methyl).

In one preferred embodiment R⁵ is a alkoxy, preferably methoxy and R², R³, R⁴, R⁶ are all H.

In another embodiment R⁵ is cyano and R², R³, R⁴ and R⁶ are H.

In the compounds of the formula I, X is H. It is believed that, hydroxylation of the compound will occur in situ at the carbon atom to which X is attached, thereby activating the compound enabling it to act as a DNA alkylating agent.

Many of the compounds of the general formula I and salts thereof are believed to be novel compounds. According to a further aspect of the invention there is provided a new compound of the general formula II or a salt thereof

in which R², R³, R⁵ and R⁶ are as defined above

X¹ is H;

Y¹ is a leaving group;

R²⁰ is —R⁷, —OR⁷, —NH₂ or Ar²;

R⁷ is as defined above;

Ar² is selected from

in which B¹ is N or CR²²;

Z¹ is O, S, —CH═CH— or NR²¹;

R²¹ is an amine protecting group;

the or each R²² is selected from OH, C₁₋₄ alkoxy C₁₋₄ alkyl, NO₂, —NHR²¹, —NHR²⁶, —NR²⁶ ₂, —N⁺R²⁶ ₃, —CN, Cl, Br, I, —NHCOR²⁵, —COOH, —CONHR⁷ and —COOR²⁵;

p is an integer in the range 0 to 4;

R²³ is H, COAr³, —CONH₂, —COOH, —CONHR⁷ or —COR⁷ or is an amine protecting group;

the or each R²⁴ is selected from OH, C₁₋₄ alkoxy C₁₋₄ alkyl, NO₂, —NHR²¹, —NHR²⁶, —NR²⁶ ₂, —N⁺R²⁶ ₃, —CN, Cl, Br, I, —NHCOR², —COOH, —CONHR⁷ and —COOR⁷;

q is 0, 1 or 2;

R²⁶ is selected from C₁₋₄ alkyl; optionally substituted phenyl, optionally substituted heteroalkyl, C₇₋₁₂ aralkyl Ar³ and a ligand.

R²⁵ is selected from C₁₋₄ alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl and optionally substituted heteroaryl; and

Ar³ is selected from the same groups as Ar²

provided that no more than one R²² or R²⁴ in any one ring includes a group Ar³.

Ar³ is preferably

Compounds of the formula II or IIA, in which primary or secondary amine nitrogen atoms are protected are generally deprotected before being used in pharmaceutical compositions. Examples of amine protecting groups R²¹ or R²³ are benzyl, benzyloxycarbonyl, tertiary butyloxycarbonyl (BOC), fluorenyl-N-methoxy-carbonyl (FMOC) and 2-[biphenylyl-(4)]-propyl-2-oxycarbonyl. Where more than one amine group is protected in the molecule, the protecting groups may be the same or different. In a particularly useful series of compounds of the general formula II and IIA, R²⁰ is OR⁷ and R⁷ is an amine protecting group different to R¹CO. In another preferred series R²⁰ is other than OR⁷.

In compounds of the general formula II and IIA, R²⁰ is preferably Ar², and/or Y¹ is preferably selected from —OCOOR¹⁷, —OCONHR¹⁸, Cl, Br, I, or —OSOOR¹⁹, in which R¹⁷, R¹⁸ and R¹⁹ are selected from C₁₋₄ alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl and optionally substituted heteroaryl. Most preferably the leaving group is a halogen atom, preferably chlorine.

The present invention further provides pharmaceutical compositions comprising compounds of the formula I or IA and a pharmaceutically acceptable excipient. Pharmaceutical compositions may be suitable for intramuscular, intraperitoneal, intrapulmonary, oral or, most preferably, intravenous administration. The compositions contain suitable matrixes, for example for controlled or delayed release. The compositions may be in the form of solutions, solids, for instance powders, tablets or implants, and may comprise the compound of the formula I in solid or dissolved form. The compound may be incorporated in a particulate drug delivery system, for instance in a liquid formulation. Specific examples of suitable excipients include lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate. Solid compositions may take the form of powders and gels but are more conveniently of a formed type, for example as tablets, cachets or capsules (including spansules). Alternative, more specialised types of formulation including liposomes, nanosomes and nanoparticles.

Compounds of the formula I may be synthesised using techniques analogous to those summarised by Boger et al 1997, op. cit. It is convenient to form the DNA alkylating sub unit in one series of steps and to attach this is through the nitrogen atom of the dihydro-pyrrole or tetrahydroquinoline (C) ring to the rest of the molecule. The DNA alkylating sub-unit may be conjugated to DNA binding sub-units synthesised as described in Boger et al, 1997 op. cit., for instance the PDE-1 and PDE-II sub-units described in that reference. The DNA binding subunits are those including the groups Ar and Ar¹.

According to a further aspect of the invention there is provided a new synthetic method in which a compound of the formula III or IIIA

in which R², R³, R⁴, R⁵ and R⁶ are as defined above;

X² is H; and

Y² is a leaving group or a hydroxyl or protected hydroxyl group; is reacted with a compound of the general formula IV R²⁷COY³  IV

in which R²⁷ is selected from C₁₋₄-alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl and Ar⁴;

Ar⁴ is selected from

in which B² is N or CR³²;

Z² is O, S, —CH═CH— or NR³³;

the or each R²⁸ is selected from C₁₋₄-alkoxy, C₁₋₄-alkyl, NO₂, CN, Cl, Br, I, —NHR³³, —NHR³⁵, —NR³⁵ ₂, —N⁺R³⁵ ₃—, —NHCOR³⁴, —COOH, —CONHR³⁶ and —COR³⁶;

r is an integer in the range 0 to 4;

R²⁹ is an amine protecting group;

R³⁰ is an amine protecting group, —CONH₂, —COOH, —COR³⁶ or —COAr⁵;

the or each R³¹ is selected from C₁₋₄-alkoxy, C₁₋₄-alkyl, NO₂, CN, Cl, Br, I, —NHR³³, —NHR³⁵, —NR³⁵ ₂, —N⁺R³⁵ ₃—, NHCOR³⁴, —COOH, —CONHR³⁶ and —COOR³⁶;

s is 0, 1 or 2;

R³² is selected from H, C₁₋₄-alkoxy, C₁₋₄-alkyl, NO₂, CN, Cl, Br, I, —NHR⁵³, —NHR³⁵, —NHR³⁵ ₂, —N^(T)R³⁵ ₃, NHCOR³⁴, COOH, —CONHR³⁶ and COOR³⁶;

R³³ is an amine protecting group;

R³⁴ is selected from Ar⁵, C₁₋₄-alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl and a ligand;

R³⁵ is selected from C₁₋₄-alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl and optionally substituted heteroaryl;

R³⁶ is selected from C₁₋₄-alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl optionally substituted heteroaryl and a ligand;

Ar⁵ is selected from the same groups as Ar⁴ and

Y³ is a leaving group

provided that no more than one R²⁸ or R³¹ in any one ring includes a group Ar⁵.

A⁵ is preferably

Y³ is, for instance, selected amongst the preferred leaving groups listed above for Y. Most suitably the definition of Y³ is Cl. Alternatively, the group Y³ may be OH. In this case, it may be necessary to include a coupling agent to assist in the coupling reaction.

The reaction between the compound of the general formula III or IIIA and the carboxylic acid or derivative of the general formula IV is carried out under conditions allowing such coupling to take place. Such conditions are similar to those generally used for formation of peptide bonds, for instance as used in peptide synthetic methods.

Y² is a hydroxy or protected hydroxyl group or a leaving group, which may be the same as Y, or may be converted to Y in a subsequent step.

After the coupling process, it may be desirable to deprotect one or more of the protected amine groups. If further reaction, for instance with other derivatising agents such as glycosyl compounds, peptides, polymers etc is desired through any such amine groups, it may be desirable to deprotect only those to which subsequent reaction to take place, whilst retaining the other amine groups in a protected form. Selection of suitable amine protecting groups and protection and deprotection protocols may be made using techniques commonly utilised in peptide chemistry.

It is believed that some of the intermediates of the general formula III or IIIA may be novel compounds. A novel compound may have the general formula V or VA

in which R³⁸, R³⁹, R⁴⁰, R⁴¹ and R⁴² are selected from H, C₁₋₄-alkyl, —OH, C₁₋₄-alkoxy, —CN, Cl, Br, I, NO₂, NHR⁴³, —NHCOR⁴⁴, —NR⁴⁵ ₂, —N⁺R⁴⁵ ₃, —COOH, —CONHR⁴⁶ and —COOR⁴⁶

X² is H;

Y² is a leaving group or a hydroxyl or protected hydroxyl group;

R³⁷ is H or an amine protecting group;

R⁴³ is selected from H, C₁₋₄ alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl, and optionally substituted heteroaryl an amine protecting group;

R⁴⁴ is selected from C₁₋₄ alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl and a ligand;

each R⁴⁵ is selected from C₁₋₄alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl and optionally substituted heteroaryl; and

R⁴⁶ is selected from C₁₋₄alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl and a ligand.

In compounds of the general formula III, in the compound ready for reaction with a carboxylic acid derivative, for instance of the general formula IV, R²⁴ is H. Precursors for such compounds have the ring nitrogen atom in protected form, that is in which R³⁷ represents a protecting group.

In compounds of the formula V, the group Y may be selected amongst those defined above for leaving group Y. The nature of the group Y² should be selected having regard to the nature of the reagent with which the compound of the formula III/V is to react in a subsequent step, for instance with a compound of the general formula IV, such that the group Y² is not deactivated and does not form a dimer of compounds of the formula III or V. Suitable examples of leaving group Y are Cl and Br.

The compound of the formula III or IIIA and V or VA may be prepared in a preliminary step including a cyclisation step in the presence of a catalyst using as the starting material an aniline compound having a leaving group substituent Y⁴ at the carbon atom ortho to the amine group substituent, and an N-substituent which is a group —CH₂CH═CHY⁵, in which the aniline derivative is reacted under cyclisation conditions, to form a dihydropyrrole or a di- or tetra hydroquinoline ring. The starting compound for such a reaction may be represented by the general formula VI

in which R³⁸ through R⁴² and X² are the same as in the compound of the formula IV;

R⁴⁷ is an amine protecting group,

one of Z¹ and Z² is Y⁵ and the other is H;

Y⁵ is a leaving group which is different from or the same as Y²; and

Y⁴ is the radical leaving group.

For cyclisation to form a dihydropyrrole ring, the group Z¹ is Y⁵ and Y⁵ is either H or a leaving group, preferably the same group as Y² wherein the group Y⁵ is not effective as a leaving group in this step of the synthesis. The reaction is conducted in the presence of a suitable catalyst, optionally in the presence of a free radical trap. The group Y⁴ should be a radical leaving group, such as halogen, preferably Br or I. Suitable radicals for carrying out the cyclisation reaction using a compound VI in which Y⁵ is H are nitroxy compounds such as 2,2,6,6-tetramethylpiperidinyloxy (TEMPO). Where Y⁵, is a leaving group the cyclisation may be carried out in the presence of a radical derived from azoisobutyronitrile (AIBN). Suitable catalysts for such a radical cyclisation step are tin hydride compounds such as tributyl tin hydride. Such a synthetic route is illustrated in Examples 1 and 3.

For cyclisation to form a 6-membered ring it is preferred to use a compound VI in which Z² is Y⁵ and Y⁵ is a leaving group, preferably a trialkyl stannyl group, and to carry out the reaction in the presence of a suitable catalyst palladium complexes such as tetrakis(triphenylphosphine) palladium (0), bis(triphenyl phosphine) palladium (II) chloride or palladium (II) acetate. The dihydroquinoline intermediate is oxidised to form a further intermediate which is an epoxide, for instance using a peroxide reagent. The epoxide intermediate is reduced using a suitable selective reducing agent such as a dialkyl aluminium hydride to produce the corresponding tetrahydroquinoline alcohol which is subsequently halogenated, for instance using carbon tetrachloride/triphenyl phosphine. This reaction is illustrated in Example 2.

The compound of the general formula VI may be produced by alkylation of the sodium salt of the corresponding aniline derivative with a 1,3-dihalo propene compound.

The carboxylic acid derivative of the general formula V may be synthesised using the methods generally described in Boger et al, 1997 op.cit. for instance PDE-I and PDE-II may be synthesised using the Umezawa synthesis, the Rees-Moody synthesis, the Magnus synthesis, the Cava-Rawal synthesis, the Boger-Coleman synthesis, the Sundberg synthesis, the Martin synthesis, the Tojo synthesis. Indole-2-carboxylic acid is commercially available. Other analogues of the DNA binding sub-units of the duocarmycins, and reactive carboxylic acid derivatives thereof are described by Boger et al, op.cit. and in U.S. Pat. No. 5,843,937.

The present invention relates to the creation of a range of prodrugs that have little or no cytotoxic effects when in their normal state, but are highly cytotoxic (i.e. have a substantially increased cytotoxicity) when activated by oxidation or hydroxylation by CYP enzymes. This provides for a self-targeting drug delivery system in which a non cytotoxic (or negligibly cytotoxic) compound can be administered to a patient, for example in a systemic manner, the compound then being activated at the site of the tumor cells (intratumoral activation) to form a highly cytotoxic compound which acts to kill the tumor cells. The fact that the CYP isoforms are not expressed by normal cells mean that the activation of the compound only occurs at the site of the tumor cells and therefore only tumor cells are affected, thus providing a self-targeting system.

The prodrugs of the present invention have the distinct advantage of being useful in the treatment of tumors at any site in the body, meaning that even tumors that have undergone metastasis (which are normally not susceptible to site specific therapies) may be treated.

The prodrug may be an antitumor prodrug. Examples of tumors include cancers (malignant neoplasms) as well as other neoplasms e.g. innocent tumors. The prodrug may be activated by hydroxylation by isoforms of cytochrome P450's.

In a variation of the normal procedure which relies upon CYP expression within tumor cells to effect selective hydroxylation and hence activation of the prodrugs, the selectivity between tumor tissue and normal tissue can be enhanced in a two part procedure. Thus (a) infecting tumor cells with a viral vector carrying a cytochrome P450 gene and a cytochrome P450 reductase gene, wherein expression of cytochrome P450 gene and cytochrome P450 reductase gene by tumor cells enables the enzymatic conversion of a chemotherapeutic agent to its cytotoxic form within the tumor, whereby the tumor cells become selectively sensitized to the prodrug chemotherapeutic agent (b) contacting tumor cells with the prodrug chemotherapeutic agent whereby tumor cells are selectively killed.

These prodrugs are benz(e)dihydroindole or benz-tetrahydroquinoline derivatives. Their specific use as antitumor prodrugs has not been previously suggested or disclosed, nor has the suggestion that they are prodrugs having an activated hydroxylated form. Where compounds of formula (I) have been previously identified and made, they have not been identified as anti-tumor agents due to their poor (or negligible) cytotoxicity. Thus the intratumoral hydroxylation of the prodrugs of the present invention provides them with a surprising and unexpected efficacy.

Hydroxylated forms of the prodrugs are potent DNA alkylating agents that bind in the minor groove of DNA and alkylate the purine bases at the N3 position. As such, they are potent cytotoxic agents whose exact biological mechanism of action is unknown but involves the disruption of template and other functions of DNA. General inhibition of template function of DNA will affect and be generally cytotoxic to all dividing cells in the body and lead to unacceptable side effects in a therapeutic setting. However, the targeted production of hydroxylated forms only in tumor cells that overexpress particular isoforms of cytochrome P450's will lead to a specific cytotoxic effect only in those cells. The non-hydroxylated forms are essentially non-toxic to all cells.

The following examples illustrate the invention.

EXAMPLE 1

1.1 N-(tert-Butyloxycarbonyl)napthylamine

A solution of 2-napthoic acid (100 mg, 0.581 mmol) in t-BuOH (33 mL) was treated with Et₃N (96 μL, 0.7 mmol) and 4 Å molecular sieves (1 g). Diphenyl phosphorylazide (0.15 ml, 0.7 mmol) was added, and the reaction mixture was warmed to reflux for 14 h. The mixture was cooled to 25° C. and the solvent removed under vacuum. The residue was dissolved in EtOAc (10 mL) and the organic phase was washed with 10% aqueous HCl (3×15 mL), dried (MgSO₄) and concentrated. Purification by flash chromatography (SiO₂, 10% EtOAc in hexanes) afforded 2 (0.108 mg, 77%) as a pale yellow solid. FABMS (NBA/NaI) 243([M+H]⁺ expected 243) 269 ([M+Na]⁺ expected 269).

1.2 2-Napthylamine

Compound 1.1 (1.5 g, 6.17 mmol) was dissolved in EtOAc (10 mL) to which was added 2.5 M HCl in EtOAc. The reaction mixture was stirred for 30 min. The resulting solution was then diluted with sat'd aq. NaHCO₃ (50 mL) and extracted with EtOAc (2×25 mL). The combined organic layers were dried (MgSO₄) and concentrated. The residue was crystallised from 20% EtOAc in hexane to afford 3.3 (0.74 g, 84%) as pale brown crystals: ¹H NMR (250 MHz, CDCl₃) δ ppm 7.12-7.32 (m, 3H), 6.96 (t, 1H), 6.80 (t, 1H), 6.56 (s, 1H), 6.52 (dd, 1H), 3.46 (br s, 2H); FAB MS (NBA/NaI) 143 ([M+H]⁺ expected 143), 165 ([M+Na]⁺ expected 165).

1.3 1-Bromo-2-napthylamine

A solution of 1.2 (100 mg, 0.7 mmol), TsOH (48 mg, 0.28 mmol) in THF (6 mL) was stirred and cooled to 0° C. To the resulting mixture NIS (125 mg, 0.56 mmol) and THF (6 mL) were added and the solution was allowed to warm to 25° C. After 3 h, additional NIS (31 mg, 0.14 mmol) was added. After 1 h, the mixture was diluted with 10% aqueous NaHCO₃ (10 mL) and extracted with CHCl₃ (3×10 mL). The organic layers were combined, dried (MgSO₄) and concentrated. The residue was purified by flash chromatography (SiO₂, 30% EtOAc in hexane) to afford 1.3 (110 mg, 59%) as a brown-yellow oil ¹H NMR (250 MHz, CDCl₃) δ ppm 8.04 (d, 1H), 7.68 (d, 1H), 7.62 (d, 1H), 7.50 (t, 1H), 7.28 (t, 1H), 7.00 (d, 1H), 4.40 (br s, 2H); FABMS (NBA/NaI): 221 ([M+H]⁺ expected 221), 243 ([M+Na]⁺ expected 243).

1.4 N-Di(tert-butyloxycarbonyl)-1-bromo-2-napthylamine

A solution of 1.3 (100 mg, 0.37 mmol) in CH₂Cl₂ (5 mL) was treated with Boc-dicarbonate (219 mg, 1.0 mmol), Et₃N (62 μl, 0.45 mmol) and DMAP (4.5 mg, 0.037 mmol). The reaction was refluxed at 50° C. for 24 h. The resulting mixture was washed with H₂O (2×10 mL), 5% HCl (10 mL) and finally again with H₂O (10 mL). The organic layers were combined, dried (MgSO₄) and concentrated. The reaction was purified by flash chromatography (SiO₂CH₂Cl₂/hexane 1:1) to afford 1.4 (125 mg, 72%) as a colourless solid. ¹H NMR (250 MHz, CDCl₃) δ ppm 8.32 (d, 1H), 7.86 (d, 1H), 7.80 (d, 1H), 7.52-7.68 (m, 2H), 7.32 (d, 1H), 1.50 (s, 18H); FABMS (NBA/NaI) 422 ([M+H]⁺ expected 422), 446 ([M+Na]⁺ expected 446).

1.5 N-(tert-Butyloxycarbonyl)-1-bromo-2-napthylamine

Compound 1.4 (50 mg, 0.107 mmol) was treated with a solution of TFA (16 μL, 0.213 mmol) in CH₂Cl₂ (2 mL) and stirred for 45 min. The mixture was concentrated and recrystallised from hexane to afford 1.5 (13 mg, 34%) as white crystals. FABMS (NBA/NaI) 323 ([M+H]⁺ expected 323), 346 ([M+Na]⁺ expected 346).

1.6 N-(tert-Butyloxycarbonyl)-N-(3-chloro-2-propen-1-yl)-1-bromo-2-napthylamine

A solution of 1.5 (50 mg, 0.14 mmol) and DMF (5 mL) was cooled to 0° C. and NaH (9.8 mg, 0.408 mmol) was added. The resulting mixture was stirred for 15 min and 1,3 dichloropropene (38 μL, 0.41 mmol) was added. The solution was allowed to warm to 25° C. and stirred for 90 min. The mixture was concentrated and the residue was purified by flash chromatography (SiO₂, 10% EtOAc in hexane) to afford 1.6 (56 mg, 93%) as a clear film. FABMS (NBA/NaI) 396 ([M+H]⁺ expected 396), 418 ([M+Na]⁺ expected 418).

1.7 3-(tert-Butyloxycarbonyl)-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indole

A solution of 1.6 (55 mg, 0.12 mmol) and AIBN (8 mg, 0.05 mmol) in anhydrous toluene (5 mL) was degassed for 15 min with N₂ and then heated to 90° C. Bu₃SnH (65 μl, 0.25 mmol) was added in four portions over 1 h and the resulting mixture was stirred for 1 h. The solution was then concentrated and purified by flash chromatography (SiO₂, 10% EtOAc in hexane) to afford impure 3.8. The solid was then dissolved in EtOAc (2 mL) to which a 1M KF solution (1 ml) in EtOAc (1 ml) was added. The solution was stirred for 45 mins after which the insoluble precipitate was filtered and the remaining solution concentrated to afford pure 1.7 (34 mg, 87%) as a pale yellow oily film. FABMS (NBA/NaI) 317 ([M+H]⁺ expected 317).

1.8 1-(Chloromethyl)-3-[(5-methoxyindol-2-yl)carbonyl]-1,2-dihydro-3H-benz[e]indole

Compound 1.7 (10 mg, 0.03 mmol) was treated with 2.5 M HCl in EtOAc (100 μL) and the solution was stirred for 30 min. The solvent was removed under a stream of nitrogen and the grey residue was dissolved in DMF (1 mL). 5-Methoxyindole-2-carboxylic acid (17 mg, 0.09 mmol) and EDC (17 mg, 0.09 mmol) were added and the mixture stirred for 16 h. Solvent was removed in vacuo and the residue subjected to flash chromatography (SiO₂, EtOAc/hexanes 1:1) to give the product as a red oil (11 mg, 94%). FABMS (NBA/NaI) 391 (M+H⁺ expected 391).

EXAMPLE 2

2.1 2-[N-(3-(tributylstannyl)-2-propen-1-yl) -N-((tert-butyloxy) carbonyl)]amino-1-napthalene

1-benzoyl-5-(tert-butyloxycarbonyl)amino-4-iodoindole (100 mg, 0.22 mmol) was stirred in DMF (1 mL) and sodium hydride (26 mg, 0.66 mmol, 60% dispersion in oil, 3 equiv.) was added. After 15 min, the suspension was treated with E/Z-1-tributylstannyl-3-bromopropene (270 mg, 0.66 mmol, 3 equiv) and the resulting solution was stirred at RT for 16 h. The solution was concentrated and water (10 mL) was added. The aqueous solution was extracted with ethyl acetate (3×10 mL), the organic layers combined, dried and concentrated. Flash chromatography (SiO₂, 10% ethyl acetate/hexanes) gave the product (120 mg, 78%) as a yellow solid. FABMS (NBA/NaI): 699 (M+H⁺ expected 699).

2.2 1,2-dihydro-1-((tert-butyloxy)carbonyl)-5,6-benzoquinoline

1-Benzoyl-5-[N-(3-(tributylstannyl)-2-propen-1-yl) -N-((tert-butyloxy)carbonyl)]amino-4-iodoindole (100 mg, 0.14 mmol) and tetrakis(triphenylphosphine) palladium(0) (32 mg, 0.2 equiv) were stirred in toluene (2 mL) at 50° C. under N₂ for 4 h. The solvent was then removed in vacuo. Chromatography (SiO₂, 10% ethyl acetate/hexanes) gave the product as a red oil (38 mg, 99%). FABMS (NBA/NaI): 282 (M+H⁺ expected 282).

2.3 3,4-epoxy-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-benzoquinoline

1,2-dihydro-1-((tert-butyloxy)carbonyl)-5,6-benzoquinoline (100 mg, 0.36 mmol) and MCPBA (91 mg, 0.54 mmol, 1.5 equiv) were stirred in CH₂Cl₂ (2 mL) at −30° C. under N₂ for 6 h. The solvent was then removed in vacuo. Chromatography (SiO₂, 10% ethyl acetate/hexanes) gave the product (101 mg, 95%). FABMS (NBA/NaI): 298 (M+H⁺ expected 298).

2.4 4-hydroxy-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-benzoquinoline

3,4-epoxy-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-benzoquinoline. (100 mg, 0.34 mmol) was treated with Dibal-H (91 mg, 0.54 mmol, 1.5 equiv) in THF (2 mL) at −78° C. to −30° C. under N₂. After 1 h, the reaction was quenched by the addition of water (2 mL) and the resulting solution was extracted with ethyl acetate (3×10 mL), the organic layers combined, dried and concentrated. The solvent was removed in vacuo. Chromatography (SiO₂, 10% ethyl acetate/hexanes) gave the product (55 mg, 54%) as a colourless solid. FABMS (NBA/NaI): 300 (M+H⁺ expected 300), 322 (M+Na⁺ expected 322).

2.5 4-Chloro-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-benzoquinoline

4-hydroxy-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-benzoquinoline (100 mg, 0.33 mmol) in CH₂Cl₂ (2 mL) was treated with a prepared solution of PPh₃ (175 mg, 0.66 mmol, 2 equiv) and CCl₄ (200 μL) in CH₂Cl₂ (2 mL) at RT. After 4 h, the solvent was removed in vacuo. Chromatography (Silica gel, 2×15 cm, 10% ethyl acetate/hexanes) gave the chloride as an oil (65 mg, 63%). FABMS (NBA/NaI): 318 (M+H⁺ expected 318), 340 (M+Na⁺ expected 340). The product may subsequently be deprotected and conjugated to a DNA-binding subunit such as 5-methoxyindole-2-carboxylic acid by process steps analogous to example 1.8.

EXAMPLE 3 BIOLOGICAL TESTING OF COMPOUND 1.8 Materials and Methods 3.1 Incubation Mixtures of Test Compound and Microsomes

Test compound activation by CYP enzymes was carried out using NADPH supplemented rat liver microsomes. Incubation mixtures comprised microsomal protein (1 mg/ml), reduced-nicotinamide adenine dinucleotide phosphate (NADPH, 10 mM) and phosphate buffer (pH7.4, 100 mM). Test compound (0.01-100 μM final concentration) in DMSO (201) was added to the microsomal incubation mixtures (0.5 ml) and incubated for 60 min at 37 C. Control incubates contained test compound and microsomal incubation mixture terminated at 0 time. All incubations were terminated by addition of an equal volume of ice-cold acetonitrile and microfuged for 3 min. Aliquots of the supernatant were added to cells in culture and cytotoxicity determined as described below.

3.2 Cell Culture Based Cytotoxicity Measurement

Chinese Hamster Ovary (CHO) cell were grown in MEM supplemented with 10% dialysed FBS and G418 (400 μg/ml). All cells were seeded at an initial density of 1000 cells/well in 96-well-plates, incubation at 37° C. for 24 hours. Aliquots (0.1 ml) of the test compound/microsomal/acetonitrile supernatant was then added to the CHO cells. Cells were then incubated for 24 hours at 37° C., 5% CO₂. After this time period MTT (50 μl; 2 mg/ml stock solution) was added to each well and cells were incubated for a further 4 hours. During this time period MTT, a hydrogen acceptor tetrazolium salt, is reduced to formazan dye by mitochondrial dehydrogenase of viable cells. The media was aspirated from cells and DMSO (100 μl/well) added to solubilise the coloured formazan dye. Absorbance of the formazan dye in the 96-well-plates was then determined at 550 nm. The effect of microsomal activation by the test compound on the arrest of CHO cell growth could be determined by comparing the IC₅₀ (concentration that inhibited cell growth by 50%) with and without microsomal incubation.

Results

CHO IC50 (μM) compound +activation −activation AF 1.8 0.1 ± 1.2 1.5 ± 0.53 15.0* *represents significance at p > 0.05.

Effect of compound 1.8 and its metabolism (activation) product on the survival of Chinese hamster ovary cells in culture. Cells were incubated for 24 hours with supernatants from reaction mixtures of compound 1.8 with NADPH fortified rat liver microsomes. IC₅₀ represents the concentration of drug required to inhibit cell growth by 50%. Values are expressed as the mean+sd for three experiments. See methods for full details of metabolism. AF=activity factor i.e. the ratio of IC₅₀ cytotoxicity values obtained for +compound 1.8 activation.

-   -   * represents significance at p>0.05. 

1. A method of inhibiting the growth of tumor cells expressing a cytochrome P450 enzyme comprising contacting the tumor cells with:

in which X is H; Y is selected from the group consisting of —OCOOR¹⁷, —OCONHR¹⁸, Cl , Br, I, and —OSOOR¹⁹ in which R¹⁷, R¹⁸ and R¹⁹ are selected from the group consisting of C₁₋₄ alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl and optionally substituted heteroaryl; R¹ is —Ar; R², R³, R⁴, R⁵ and R⁶ are each independently selected from the group consisting of H, C₁₋₄ alkyl, —OH, C₁₋₄ alkoxy, —CN, Cl, Br, I, —NO₂, —NH₂, —NHR¹⁶, —NR¹⁶ ₂, —N⁺R¹⁶ ₃, —NHCOR⁸, —COOH, —CONHR⁹, —NHCOOR⁹ and —COOR⁹; R⁸ and R⁹ are independently selected from the group consisting of C₁₋₄ alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl and a ligand; Ar is selected from the group consisting of:

in which B is N or CR¹⁰; R¹⁰ is selected from the group consisting of OH, C₁₋₄ alkoxy, C₁₋₄ alkyl, —NO₂, —NH₂, —CN, Cl, Br, I, —NHCOR¹⁴, —COON, —CONHR¹⁵, —NHCOOR¹⁵, —COOR¹⁵ and H; Z is selected from the group consisting of O, S, —CH═CH—and NH; the or each R¹¹ is selected from the group consisting of OH, C₁₋₄ alkoxy, C₁₋₄ alkyl, —NO₂, —NH₂, —NHR¹⁶, —NR¹⁶ ₂, —N⁺R¹⁶ ₃, —CN, Cl, Br, I, —NHCOR¹⁴, —COOH, —CONHR¹⁵ —NHCOOR¹⁵ and COOR¹⁵; n is an integer in the range 0 to 4; R¹² is selected from the group consisting of H, —COAr¹, —CONH₂, —COOH, —COR¹⁵ or —COOR¹⁵; the or each R¹³ is selected from the group consisting of OH, C₁₋₄ alkoxy, C₁₋₄ alkyl, —NO₂, —NH₂, —NHR¹⁶, —NR¹⁶ ₂, —N⁺R¹⁶ ₃, —CN, Cl, Br, I, —NHCOR¹⁴, —COOH, —CONHR¹⁵, —NHCOOR¹⁵ and —COOR¹⁵; m is 0, 1 or 2; R¹⁴ is selected from the group consisting of C₁₋₄ alkyl, optionally substituted phenyl, optionally substituted heteroaryl, C₇₋₁₂ aralkyl, Ar¹ and a ligand; R¹⁵ is selected from the group consisting of C₁₋₄ alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl and optionally substituted heteroaryl and a ligand; the or each R¹⁶ is independently selected from the group consisting of C₁₋₄ alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl and optionally substituted heteroaryl; and Ar¹ is selected from the same groups as Ar; provided that no more than one group R¹¹ or R¹³ in any one ring includes a group Ar¹.
 2. A method according to claim 1 in which R³ is H.
 3. A method according to claim 2 in which R⁴ is H.
 4. A method according to claim 3 in which R⁵ is H.
 5. A method according to claim 4 in which —Ar is


6. A method according to claim 5 in which n is at least 1 and one of the groups R¹¹ of the —Ar group is —NHCOAr¹.
 7. A method according to claim 6 in which B is N.
 8. A method according to claim 6 in which B is CR¹⁰.
 9. A method according to claim 8 in which R¹⁰ is H.
 10. A method according to claim 9 in which Y is Cl.
 11. A method according to claim 10 in which Z is NH.
 12. A method according to claim 11 in which R¹¹ is methoxy and n is
 1. 13. A method according to claim 5 in which B is N.
 14. A method according to claim 5 in which B is CR¹⁰.
 15. A method according to claim 14 in which R¹⁰ is H.
 16. A method according to claim 5 in which Y is Cl.
 17. A method according to claim 16 in which R² is H.
 18. A method according to claim 4 in which Ar is


19. A method according to claim 17 in which R¹² is —COAr¹.
 20. A method according to claim 1 wherein the cytochrome P450 enzyme is a CYP1B1 enzyme. 